The unique and complex architecture of the Golgi apparatus has been conserved through most of eukaryotic evolution but the role it plays in the functioning of this organelle is far from clear. One reason has been the lack of knowledge concerning those structural proteins responsible for generating and maintaining the central architectural feature of the Golgi apparatus, the stacks of closely-apposed and flattened cisternal membranes with associated transport vesicles. By exploiting the dramatic transformation of the Golgi during mitosis in animal cells, we have identified a complex of two proteins that play a crucial role in Golgi architecture. GRASP65 is involved in stacking cisternae whereas GM130 mediates the docking (or tethering) or transport vesicles with cisternal rims. Our aim is to work our precisely how GRASP65 and GM130 carry out their functions and to identify other proteins that help them determine Golgi structure. These proteins will also be used as tools to manipulate Golgi architecture and so help us understand its relationship to Golgi function. The specific aims of this proposal are: 1. To work out how GRASP65 stacks cisternae and whether additional proteins are required. If GRASP65 only stacks a subset of Golgi cisternae then the stacking mechanism of other cisternae will be studied. 2. To disrupt the architecture of the Golgi in vivo using GRASP65 antibodies and fragments as tools and to determine the effects on a variety of Golgi functions. 3. To elucidate the mechanism of vesicle tethering and the roles played by GM130, Giantin and p115. Biochemical and microscopic approaches will be used to work out the sequence of events that assemble and disassemble tethering complexes and to identify and characterise those GTPases and kinases that regulate this process. 4. To determine whether a template is needed for the reassembly of the Golgi at the end of mitosis and during biogenesis by studying cytoplasts that lack a Golgi apparatus.